ABSTRACT Every day, about 400 infants acquire HIV-1 globally, the vast majority by breast milk transmission. Late HIV-1 diagnosis in pregnancy, lack of adherence, and acute HIV-1 infection during the breastfeeding period remain significant hurdles in the prevention of mother-to-child-transmission (MTCT). Thus, to achieve the goal of an AIDS-free generation, we need preventive measures in addition to antiretroviral therapy (ART). An HIV vaccine represents a core component of these efforts. Our long-term goal is the development of a pediatric HIV vaccine to protect against breastmilk transmission of HIV. Despite HIV being transmitted primarily by mucosal routes, few vaccine strategies explored mucosal routes of immunization to induce protective immune responses at potential mucosal entry sites, including the oral mucosa in infants. To close these gaps, we started to explore the potential of oral vaccination in the protection against breastmilk transmission of HIV. Newborn macaques vaccinated with a combined oral (PO) and intramuscular (IM) DNA-SIV prime and boosted by combined sublingual (SL) and IM MVA-SIV had a significantly reduced per-exposure-risk of oral SIV infection compared to infants receiving the same vaccine by the IM route alone. Reduced infection risk was associated with higher SIV Env gp120 and V1V2 specific IgG antibodies in fecal specimens. Based on this premise, we present the central hypothesis that a rationally designed combined SL+parental vaccine regimen can prevent oral HIV acquisition in infants. The rich regional lymphatic network of the Waldeyer?s Ring provides an easy and non- invasive portal for oral vaccine uptake, and, as an intrinsic part of the systemic lymphatic network, enables the induction of local and systemic protective immune responses by oral vaccines. The objective of the proposed studies is to optimize our pediatric vaccine through rational selection of adjuvants and HIV Env immunogens that can induce Env-specific IgG and IgA antibodies in plasma and at mucosal entry sites, such as the oral mucosa and the intestine. We will immunize infants by the SL and subcutaneous (SC) route with MVA expressing SIVgag, pol and the clade C HIV C.1086 envelope (Env) NFL trimer plus Env NFL trimer protein and test the hypothesis that a combined SL+SC vaccine regimen provides superior efficacy against oral SHIV CH848 challenge compared to vaccination by only the SC route (Aims 1 and 2). Both Env immunogen and challenge virus contain Envs of HIV clade C, the clade most prevalent in sub-Saharan Africa where the majority of pediatric HIV infections occur, emphasizing the clinical relevance of our studies. We further test novel adjuvants that were selected to specifically enhance mucosal immune responses and the priming function of infant dendritic cells. To identify pathways that are essential for the generation of highly functional antibody responses in infants and are associated with protection against oral SHIV CH848 challenge in infant macaques, we will define oral innate molecular signatures that determine vaccine-induced adaptive immune responses at the time of challenge and are correlated with challenge outcome (Aim 3).